The overall goal of this PPG proposal is to advance fundamental understanding of grp94, a major oncogenic chaperone in the endoplasmic reticulum (ER), with the ultimate objective of developing rational grp94-based cancer therapeutics. Also known as gp96, endoplasmin, and Hsp90b1, grp94 is the recently evolved ERresident member of the HSP90 family. Its expression is upregulated by the unfolded protein response that is characteristic of cancer cells. Over-expression of grp94 in cancers uniformly correlates with advanced stage and poor survival. Four recent publications from this PPG group have further cemented the oncogenic roles of grp94. Despite its high relevance in cancer, the understanding of the function and mechanism of grp94 has lagged behind that of other molecular chaperones. This has now begun to change, with several key observations with regard to client repertoire, cellular function, disease implications, structural biology, and speciic inhibitors coming from the laboratories of this PPG team. This team has shown that grp94 is an essential master chaperone for Toll-like receptors (TLRs), integrins, Wnt co-receptor LRP6, HER2 and TGF?- surface docking molecule GARP. The chaperone function of grp94 depends on its ATPase domain, which has been structurally resolved by the team and shown to be distinct from that of cytosolic Hsp90, enabling the successful development of several highly selective grp94 inhibitors. Two pressing tasks impede further advances in this field: 1) understanding the fundamental roles of grp94 in cancer biology; and 2) developing and validating effective grp94 inhibitors for development and clinical translation as grp94-based cancer therapeutics. This Program Project is designed to overcome both obstacles by uniting, in three Projects and two Cores, three leading laboratories in the field of grp94 research: biology (Li), structural biology (Gewirth) and drug discovery (Chiosis). This collaboration synergizes resources from three leading institutes -MUSC, Memorial Sloan-Kettering and Hauptman-Woodward - and builds on the Project Leaders' inherent shared interests, proven track records (57 joint publications) and complementary expertise. The specific goals of this PPG include: Project 1) Determine the role and significance of grp94 in controlling TGF? biogenesis and TGF?-mediated cancer progression via folding GARP and integrins; Project 2) Develop chemical tools that enable a spatio-temporal investigation of grp94-regulated cancer mechanisms in cancer phenotypes; Project 3) Elucidate the chaperone mechanism of grp94 as well as grp94 inhibitors from the structural point-of-view. The success of these projects will significantly advance understanding of how grp94 functions in cancer biology molecularly, why 'grp94-addiction' by cancer appears to span a wide spectrum of cancer types, how grp94 differs structurally and functionally from other HSP90 paralogs in binding to ATP and purine scaffold inhibitors, what are the ideal cancer types for grp94-targeted therapy, and whether a novel class of highly selective grp94 inhibitors can be used to probe the tumor biology and biological significance of grp94-client networks in cancer.